This study examined the effects of different drying methods on the chemical content, antioxidant, antibacterial, enzymatic and anticancer activity values of stevia (Stevia rebaudiana Bert.) extract. Stevia leaves were dried using four different methods (in the sun, shade, air conditioner, and oven), and extracts were collected and analyzed. Based on extract content, 2-tetradecyl acrylate was the major ingredient in air-conditioned and oven-dried applications (25.00% and 21.47%, respectively). The same compound was detected in both sun-drying and shade-drying methods, but the content was low. The antioxidant activity values of the samples were evaluated with 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging tests. While the best result for DPPH (IC50 value 57.94 ± 0.63 µg/mL) was found in the shade-dried stevia samples, the best result in terms of ABTS test (IC50 value 44.03 ± 1.22 µg/mL) ml) was detected in oven-dried. When the samples were examined in terms of antimicrobial activity, it was seen that extracts of all drying methods were effective against Staphylococcus aureus. However, it was determined that the extracts obtained from plants dried in air conditioning and oven had a stronger effect. The cell viability assay was utilized to assess the antiproliferative effects of extract on L929 and MDA-MB-231 cell lines. The extracts did not significantly affect the L929 cell viability, while MDA-MB-231 remarkably reduced cell viability (sun drying, IC50 = 0.9 mg/mL; oven drying, IC50 = 0.65 mg/mL; shade drying, IC50 = 0.74 mg/mL; air conditioner drying, IC50 = 0.47 mg/mL). In particular, the extract obtained by the air conditioner drying method showed the most prominent cytotoxic effect. The results showed that drying using different methods had an impact on the quality standards of the stevia leaves.